Cell permeable bioactive peptide conjugates having a first bioactive peptide region coupled to a second transport peptide region allowing transfer of the first bioactive peptide region and the second transport peptide region across biological membranes to enter intact living cells for regulation of biological responses.
Various proteins and protein fragments have been shown to regulate biological responses which are of either therapeutic or research interest, or both. However, a substantial problem in utilizing these proteins, protein fragments, or chemically synthesized peptides to regulate the corresponding intracellular processes can be the level of biological availability, if any, at target receptors in the living cell, whether in-vitro or in-vivo. This lack of biological availability may be due to insolubility, a binding affinity to surrounding substrates that is greater than to the target cell receptor, instability with respect to cleavage, or with respect to modification of the peptide backbone, N-terminus, C-terminus, side chain, or other peptide or chemical moiety.
With respect to proteins or peptides such as insulin or humanin, instability with respect to cleavage or modification can to an extent be overcome by administration by injection into or proximate to the tissue which presents the corresponding binding receptors of the protein or peptide, or by introduction by injection into the circulatory system to be carried to the tissue(s) which present the corresponding binding receptors to the protein or peptide, as shown by the following examples:
Humanin has recently been shown to provide protection against forms of Alzheimer's Disease. Humanin is a 24 amino acid linear polypeptide with a single cysteine at position 8. As with insulin, humanin is rapidly degraded by gastrointestinal enzymes, and thus administration by injection may be the only presently available route to deliver the peptide. “A rescue factor abolishing neuronal cell death by wide spectrum of familial Alzheimer's disease genes and Aβ” Y. Hashimoto, T. Niikura, H. Tajima, T. Yasukawa, H. Sudo, Y. Ito, Y. Kita, M. Kawasumi, K. Koumaya, M. Doyu, G. Sobue, T. Koide, S. Tsuji, J. Lang, K. Kurokawa, and I. Nishimoto, Proc. Natl. Acad. Sci. U.S.A., 98, 6336 (2001), hereby incorporated by reference herein.
Apelin peptides are now known to be endogenous ligands at the orphan G-protein coupled receptor, APJ. Apelin and its receptor have been found in the brainstem and shown to have a role in haemodynamic homeostasis. Apelin microinjections into the nucleus tractus solitarius (“NTS”) can result in either apnea or decreased phrenic nerve discharge amplitude by up to 30%. Increases of 10-20 mm Hg in arterial pressure can also evoked from microinjection of Apelin into either of the NTS and the RVLM. “Site-specific effects of apelin-13 in the rat medulla oblongata on arterial pressure and respiration” Auton Neurosci. 101(1-2):32-8 (2002), hereby incorporated by reference herein.
In both rodents and humans, ghrelin functions to increase hunger though its action on hypothalamic feeding centers. This makes sense relative to increasing plasma ghrelin concentrations observed during fasting. Additionally, humans injected with ghrelin reported sensations of intense hunger. Ghrelin also appears to suppress fat utilization in adipose tissue, which is somewhat paradoxical considering that growth hormone has the opposite effect. Overall, ghrelin seems to be one of several hormonal signals that communicates the state of energy balance in the body to the brain. “Ghrelin causes hyperphagia and obesity in rats”, A. M. Wren et al. Diabetes 50(11):2540-72001, hereby incorporated by reference herein.
Peptide YY(3-36) (PYY(3-36)), a Y2R agonist, is released from the gastrointestinal tract postprandially in proportion to the calorie content of a meal. Peripheral injection of PYY(3-36) in rats inhibits food intake and reduces weight gain. PYY(3-36) also inhibits food intake in mice but not in Y2r-null mice, which suggests that the anorectic effect requires the Y2R. Peripheral administration of PYY(3-36) increases c-Fos immunoreactivity in the arcuate nucleus and decreases hypothalamic Npy messenger RNA. Intra-arcuate injection of PYY(3-36) inhibits food intake. PYY(3-36) also inhibits electrical activity of NPY nerve terminals, thus activating adjacent pro-opiomelanocortin (POMC) neurons. In humans, infusion of normal postprandial concentrations of PYY(3-36) significantly decreases appetite and reduces food intake by 33% over 24 h. Thus, postprandial elevation of PYY(3-36) may act through the arcuate nucleus Y2R to inhibit feeding in a gut-hypothalamic pathway. “Gut hormone PYY(3-36) physiologically inhibits food intake”, R. L. Batterham et al. Nature 418(6898):650-654 (2002), hereby incorporated by reference herein.
Intracerebroventricular injections of alpha-MSH in diet-induced obese rats results in significantly enhanced nocturnal inhibitory feeding responses. Significantly greater inhibition of nocturnal feeding by alpha-MSH and reduction in PVN alpha-MSH peptide level, suggests melanocortinergic signalling may be reduced in obesity which may account for the hyperphagia of these animals when presented with a palatable diet. “Enhanced inhibitory feeding response to alpha-melanocyte stimulating hormone in the diet-induced obese rat”, M. J. Hansen, M. J. Ball and M. J. Morris, Brain Res. 892(1):130-137 (2001), hereby incorporated by reference herein.
Xenografts of MDA-MB-468 human breast carcinoma injected subcutaneously for 6 weeks with Bomesin peptides showed powerfully suppressed growth causing a complete regression of 2 tumors. “Inhibition of growth of MDA-MB-468 estrogen-independent human breast carcinoma by bombesin/gastrin-releasing peptide antagonists RC-3095 and RC-3940-11”, Zsuzsanna et al. Cancer, Vol. 88, Issue 6, 1384-1392 (2000), hereby incorporated by reference herein.
Atrial natriuretic peptide (ANP) has the effects of a vasodilator (including the pulmonary arteries) and a physiologic diuretic. “Hemodynamic Effects of Human Atrial Natriuretic Peptide After Modified Fontan Procedure”, Takeshi Hiramatsu, MD, Yasuharu Imai, MD, Yoshinori Takanashi, MD, Kazuhiro Seo, MD, Masatsugu Terada, MD, Makoto Nakazawa, MD Ann Thorac Surg 1998; 65:761-764. Exogenous ANP administration has resulted in numerous physiological responses, including rapid natriuresis, diuresis, and reduction in arterial blood pressure. “Encapsulated transgene cells attenuate hypertension, cardiac hypertrophy and enhance renal function in Goldblatt hypertensive rats” Li-Guo Chen et al. The Journal of Gene medicine 6:786-797 (2004), hereby incorporated by reference herein.
Brain natriuretic peptide delivered by infusion can influence cardiovascular homeostasis mainly by reducing cardiac preload. administration of pharmacological doses of BNP to humans and experimental animals markedly affects systemic hemodynamics and renal function, because it is followed by a reduction of arterial pressure and PVR and an increase in diuresis and natriuresis. “Cardiovascular Effects of Brain Natriuretic Peptide in Essential Hypertension”, Giorgio La Villa; Gianni Bisi; Chiara Lazzeri; Caterina Fronzaroli; Laura Stefani; Giuseppe Barletta; Riccarda Del Bene; Gianni Messeri; Gaetano Strazzulla; Franco Franchi, Hypertension, 25, 1053-1057 (1995), hereby incorporated by reference herein.
Intracerebroventricular injection of recombinant CART in rats inhibits normal and starvation-induced feeding and completely blocks the feeding response elicited by NPY. Peripheral leptin administration in obese mice stimulates CART mRNA expression. CART antisera increases feeding in normal rats suggesting that CART is an endogenous regulator of food intake. Kristensen, P. et al., Nature 393, 72-76 (1998), hereby incorporated by reference herein.
CNS administration of Agouti related protein decreased oxygen consumption and decreased the capacity of BAT to expend energy. This reduction in oxygen consumption leading to a reduction in energy expenditure, together with the stimulation in food intake, may be the mechanisms by which Agrp causes an increase in body weight and adiposity. “Chronic CNS administration of Agouti related protein (Agrp) reduces energy expenditure”, C J Small, Y L Liu, S A Stanley, I P Connoley, A Kennedy, M J Stock & S R Bloom, Endocrine Abstracts 3 OC46, hereby incorporated by reference herein.
Exendin (9-39) binds to but does not activate the GLP-1 receptor, and functions as a GLP-1 receptor antagonist Exendin-3, a novel peptide from Heloderma horridum venom, interacts with vasoactive intestinal peptide receptors and a newly described receptor on dispersed acini from guinea pig pancreas. Exendin (9-39) administered acutely has been employed as a GLP-1R antagonists in multiple preclinical studies and in human experiments to probe the consequences of disrupting GLP-1R activation. J Biol. Chem. 1991 Feb. 15; 266(5):2897-902, hereby incorporated by reference herein.
Neuromedin U (NMU) is a neuropeptide with potent activity on smooth muscle which was isolated first from porcine spinal cord and later from other species. It is widely distributed in the gut and central nervous system. Peripheral activities of NMU include stimulation of smooth muscle, increase of blood pressure, alteration of ion transport in the gut, control of local blood flow and regulation of adrenocortical function. NMU is expressed in the ventromedial hypothalamus in the rat brain, and its level is significantly reduced following fasting. Intracerebroventricular administration of NMU markedly suppresses food intake in rats and may indicate that NMU is involved in the central control of feeding. “Identification of Receptors for Neuromedin U and its Role in Feeding” Howard et al. Nature 406, 70-75 (Jul. 6, 2000), hereby incorporated by reference herein.
The systemic administration of synthetic hexapeptide of growth hormone-releasing peptide selectively releases growth hormone in many species including man. Growth hormone-releasing peptide directly stimulates growth hormone release by an action at the level of the pituitary. “Systemic administration of growth hormone-releasing peptide activates hypothalamic arcuate neurons”, S. L. Dickson, G. Leng and I. C. Robinson, Neuroscience. 1993 March; 53(2):303-6, hereby incorporated by reference herein.
Intravenous injection of recombinant coupling factor 6 peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. “Mitochondrial coupling factor 6 as a potent endogenous vasoconstrictor” Tomohiro Osanai, Makoto Tanaka, Takaatsu Kamada, Takao Nakano, Koki Takahashi, Satoko Okada, Kenichi Sirato, Koji Magota, Shiho Kodama, and Ken Okumura, J. Clin. Invest. 108(7) 1023-1030 (2001).
Octreotide injection provides a long-acting pharmacologic action mimicking those of the natural hormone somatostatin. Like somatostatin, octreotide suppresses LH response, gastrin, vasoactive intestinal peptide, secretin, motilin, and pancreatic polypeptide. Norvartis Pharmaceuticals Corporation, East Hanover, N.J., Publication T2002-82 (2002).
Intravenous injection of PACAP-27 can produce dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats and can produce dose-dependent increases in heart rate. “Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat” E. J. Whalen, A. K. Johnson, S. J. Lewis; European Journal of Pharmacology Vol. 365, No. 2, pages 205-215 (1999).
Intra peritoneal injection of an 8-mer peptide derived from the nonreceptor-binding domain of urokinase (A6) has been shown to have antiangiogenic and proapoptotic effects to block the progression of breast cancer in vivo. “An antiangiogenic urokinase-derived peptide combined with tamoxifen decreases tumor growth and metastasis in a syngeneic model of breast cancer” Y. Guo, A. P. Mazar, J. J. Lubrun, and S. A. Rabbani, Cancer Res. 2002 Aug. 15; 62(16):4678-84.
GLP-1 derivatives and analogues to both rodents and man have shown promising effects on food intake and body weight suggesting that such therapies constitute potential anti-obesity treatment. In the central nervous system, pre-proglucagon and hence GLP-1, GLP-2 and OXM are exclusively found in a small population of nerve cells in the nucleus of the solitary tract. These constitute a neural pathway linking the “viscero-sensory” brainstem to hypothalamic nuclei involved in energy homeostasis. Intracerebroventricular administration of all of the three derived peptides robustly decrease food intake. It is evident that central GLP-1 agonism probably in combination with GLP-2 and/or OXM agonism constitute a potential pharmacological tool to reduce food intake and may be also enhance energy expenditure. This and other aspects of the current state of the role of central pre-proglucagon in energy homeostasis are reviewed. Larsen P J, Vrang N, Tang-Christensen M. Curr Pharm Des. 2003; 9(17): 1373-82.
Calcitonin injection suppresses resorption of bone by inhibiting the activity of osteoclasts, a cell type that “digests” bone matrix, releasing calcium and phosphorus into blood. Calcitonin 8-32 can be a potent agonist at the hCTr, with similar efficacy as human calcitonin, and a potency of 11 nM. These results were confirmed in cyclic AMP assays. Responses to calcitonin and PHM-27 could be suppressed by the antagonist salmon calcitonin (8-32). In competition binding studies, salmon calcitonin (8-32), calcitonin, and PHM-27 were each able to inhibit ^1^2^5I-calcitonin from cell membranes containing transiently expressed hCTr. These results indicate that the orphan peptide PHM-27 is a potent agonist at the hCTr. Discovery of novel peptide/receptor interactions: identification of PHM-27 as a potent agonist of the human calcitonin receptor Ma, J.-N., Currier, E. A., Essex, A., Feddock, M., Spalding, T. A., Nash, N. R., Brann, M. R., Burstein, E. S., Biochemical Pharmacology, Vol. 67, No. 7.
C-peptide, a cleavage product of proinsulin to insulin processing, induces nitric oxide (NO)-mediated vasodilation upon injection. C-peptide (70 nmol/kg iv) can significantly improved coronary flow. Moreover, C-peptide enhanced basal NO release from rat aortic segments. Young L H, et al. Am J Physiol Heart Circ Physiol. 279(4):H1453-9 (2000), hereby incorporated by reference herein.
Calcitonin Gene Related Peptide is a potent hypotensive agent and a member of the Calcitonin/CGRP multigene complex. This peptide is 37 amino acids long containing one disulfide bridge. CGRP has been shown upon injection to elevate the flow of blood and enhance the contractibility of the atrium. Zaidi, M. et al., Critical Reviews in Clinical Laboratory Sciences 28, No. 2, 109 (1990), hereby incorporated by reference herein.
Melanin-concentrating hormone (MCH), a neuropeptide expressed in central and peripheral nervous systems which plays an important role in the control of feeding behaviors and energy metabolism. MCH and alpha-MSH exert opposing and antagonistic influences on feeding behavior and the stress response and may function in a coordinate manner to regulate metabolism through a novel mechanism mediated in part by an MCH receptor. Intracerebroventricular administration in rats increased food intake in a dose-dependent manner and lowered plasma glucocorticoid levels through a mechanism involving ACTH. In contrast, alpha-MSH decreased food intake and increased glucocorticoid levels. MCH, at a twofold molar excess, antagonized both actions of alpha-MSH. alpha-MSH, at a threefold molar excess, blocked the orexigenic properties of MCH. MCH did not block alpha-MSH binding or the ability of alpha-MSH to induce cAMP in cells expressing either the MC3 or MC4 receptor, the principal brain alpha-MSH receptor subtypes. These data suggest that MCH and alpha-MSH exert opposing and antagonistic influences on feeding behavior and the stress response and may function in a coordinate manner to regulate metabolism through a novel mechanism mediated in part by an MCH receptor.” “Melanin-concentrating hormone: a functional melanocortin antagonist in the hypothalamus” David S. Ludwig, Kathleen G. Mountjoy, Jeffrey B. Tatro, Jennifer A. Gillette, Robert C. Frederich, Jeffrey S. Flier, and Eleftheria Maratos-Flier Am J Physiol Endocrinol Metab 274: E627-E633, 1998, hereby incorporated by reference herein.
Another problem associated with certain peptides such as Adrenomedullin a 52-amino acid peptide hormone with structural homology to calcitonin gene-related peptide may be that administration by aerosol requires a wt/v peptide to carrier which makes the therapuetic use of the peptide prohibitively expensive. While the aerosol administration of adrenomedullin peptide may in certain instances provide long-lasting reduction in moncrotalin-induced pulmonary hypertension and pulmonary vascular resistance, the amount of peptide utilized to achieve efficacy may prohibit commercialization as a therapy. “Adrenomedullin: a smart road from pheochromocytoma to treatment of pulmonary hypertension.” M. Westphal, M. Booke and A. T. Dinh-Xuan Eur Respir J 24; 518:520 (2004).
Understandably, there appears to be a large commercial market for synthetic peptides which target binding sites as agonists or antagonists of the corresponding biological activity of the above-described naturally occurring proteins and peptides, whether in whole or in part, and which could be administered without injection, or which have enhanced biological activity as agonist or antagonists upon delivery by injection or aerosol.